MicroRNAs in the progression of prostate cancer. The main goal of this project is to investigate the role of a set of microRNAs (miRNAs) in the progression of prostate cancer. The rationale of this project is that miRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA, and our preliminary results have provided a novel concept that miRNAs and non-coding double stranded RNAs can also activate various genes. Based on these novel observations, we hypothesize that down-regulation of a set of microRNAs can inhibit tumor suppressor genes or activate oncogenes and re-expression of these miRNAs can reverse these effects thereby regulating prostate cancer progression. These hypotheses will be tested by pursuing the following three specific aims. Specific Aim # 1. To investigate the expression of a set of miRNAs in human prostate cancer tissues and also analyze whether miRNAs can regulate cell proliferation and progression using prostate cancer cell lines. Based on our preliminary data, we have identified a set of miRNAs that are significantly downregulated in prostate cancer. We will analyze the expression of these miRNAs in human prostate cancer samples. We will over-express a defined set of individual miRNAs in prostate cancer cell lines and the modulation of targeted genes will be evaluated at both the mRNA and protein levels using real-time PCR and Western analysis, respectively. The miRNAs-mediated repression of oncogenes or activation of tumor suppressor genes will be analyzed by luciferase assays using 3'UTR or 5'UTR sequence constructs, respectively. Changes in cell growth will be analyzed by monitoring proliferation, cell cycle distribution, apoptosis and in vitro invasion. Assays to be used include cell proliferation, flow cytometry, migration, clonogenic survival, in vitro invasion and TUNEL-based ELISA apoptosis assays. Specific Aim #2: To investigate the molecular mechanisms of microRNA inactivation in prostate cancer cells. We will test the hypothesis that specific miRNAs are inactivated through epigenetic pathways. Human prostate cancer tissues will be used for analysis of methylation of miRNAs. CpG methylation in putative promoter regions of miRNAs will be examined by sodium bisulfite methylation techniques and confirm by direct DNA sequencing. We will also investigate whether histone acetylation, chromatin remodeling and associated enzymes (histone deacetylases and histone acetyltransferases) play a role in controlling expression of specific miRNAs. Specific Aim #3: Investigate whether microRNAs can inhibit growth and proliferation of human xenograft prostate tumors in a nude mouse model. To validate our in vitro results, we will also use an in vivo model of mouse xenografts with human prostate cancer cells. PUBLIC HEALTH RELEVANCE: Successful completion of these experiments will demonstrate the functional role of specific microRNAs in the suppression of prostate cancer growth and also the mechanism of inactivation of these genes in prostate cancer. In the future, these results may provide better strategies for the management of prostate cancer.